Büller H A, Rings E H, Montgomery R K, Sybicki M A, Grand R J
Department of Pediatrics, Floating Hospital, New England Medical Center, Boston, Massachusetts 02111.
Pediatr Res. 1989 Sep;26(3):232-6. doi: 10.1203/00006450-198909000-00015.
To identify potential tissue-specific characteristics of intestinal glycoprotein synthesis and processing, rat intestinal lactase-phlorizin hydrolase (L-Ph) was studied after pulse-labeling of colonic explants from 5-d-old suckling rats in organ culture and the data compared to similar studies in rat jejunum. Histologic sections of 5-d-old proximal colon showed villus-like structures lined with columnar epithelial cells. Lactase and phlorizin hydrolase activities showed tissue-specific developmental patterns. Using a MAb to small intestinal L-Ph, we were able to immunoprecipitate from colon at different ages a protein that hydrolyzed lactose and phlorizin, and whose activity was not inhibited by p-chloromercuribenzoate. After pulse-labeling for 60 min and chase for 30 min, immunoprecipitated L-Ph from total homogenates of rat colonic explants appeared on fluorography of SDS-PAGE as one band of approximately 205 kD. With increasing time of chase, it took 240 min before the precursor form was converted to the intermediate form (equivalent to the 180-kD form in jejunum) and the mature form (equivalent to the 130-kD form in jejunum), although these conversions in the jejunum were observed within 60 min of chase, and only 30 min of pulse labeling. When compared on SDS-PAGE to immunoprecipitated jejunal L-Ph, the precursor form in the colon had a slightly higher apparent mol wt than the corresponding precursor form found in the endoplasmic reticulum-Golgi fraction of the jejunum. The intermediate as well as the mature L-Ph forms in the colon were also both somewhat higher in apparent molecular weight than the same bands in the microvillus membrane fraction from jejunal explants. Removal of N-linked oligosaccharides from jejunum and colonic forms of L-Ph produced bands on SDS-PAGE with identical mobility, suggesting that the proteins were the same. The data demonstrate that, in neonatal colon, enzymatically active L-Ph undergoes biosynthetic and processing events similar to those in the jejunum. During early life, colonic L-Ph may function in the salvage of lactose not absorbed in the small intestine.
为了确定肠道糖蛋白合成与加工的潜在组织特异性特征，对5日龄哺乳大鼠结肠外植体进行脉冲标记后，研究了大鼠肠道乳糖 - 根皮苷水解酶（L-Ph），并将数据与大鼠空肠的类似研究进行比较。5日龄近端结肠的组织学切片显示，绒毛状结构内衬柱状上皮细胞。乳糖酶和根皮苷水解酶活性呈现组织特异性发育模式。使用针对小肠L-Ph的单克隆抗体，我们能够从不同年龄的结肠中免疫沉淀出一种水解乳糖和根皮苷的蛋白质，其活性不受对氯汞苯甲酸的抑制。在脉冲标记60分钟并追踪30分钟后，大鼠结肠外植体总匀浆中免疫沉淀的L-Ph在SDS-PAGE荧光自显影片上呈现为一条约205 kD的条带。随着追踪时间的增加，前体形式转化为中间形式（相当于空肠中的180-kD形式）和成熟形式（相当于空肠中的130-kD形式）需要240分钟，尽管在空肠中这些转化在追踪60分钟内即可观察到，且脉冲标记仅30分钟。在SDS-PAGE上与免疫沉淀的空肠L-Ph相比，结肠中的前体形式的表观分子量略高于空肠内质网 - 高尔基体部分中相应的前体形式。结肠中的中间形式以及成熟的L-Ph形式在表观分子量上也比空肠外植体微绒毛膜部分中的相同条带略高。从空肠和结肠形式的L-Ph中去除N-连接寡糖后，在SDS-PAGE上产生了迁移率相同的条带，表明这些蛋白质是相同的。数据表明，在新生结肠中，具有酶活性的L-Ph经历了与空肠中相似的生物合成和加工过程。在生命早期，结肠L-Ph可能在挽救小肠中未吸收的乳糖方面发挥作用。
